Sara E.F. Kost

The Prognostic Value of FoxP3+ Tumor-Infiltrating Lymphocytes in Cancer: A Critical Review of the Literature

CD8+ tumor-infiltrating lymphocytes (TIL) are associated with survival in a variety of cancers. A second subpopulation of TIL, defined by forkhead box protein P3 (FoxP3) expression, has been reported to inhibit tumor immunity, resulting in decreased patient survival. On the basis of this premise, several groups are attempting to deplete FoxP3+ T cells to enhance tumor immunity. However, recent studies have challenged this paradigm by showing that FoxP3+ T cells exhibit heterogeneous phenotypes and, in some cohorts, are associated with favorable prognosis. These discrepant results could arise from differences in study methodologies or the biologic properties of specific cancer types. Here, we conduct the first systematic review of the prognostic significance of FoxP3+ T cells across nonlymphoid cancers (58 studies from 16 cancers). We assessed antibody specificity, cell-scoring strategy, multivariate modeling, use of single compared with multiple markers, and tumor site. Two factors proved important. First, when FoxP3 was combined with one additional marker, double-positive T cells were generally associated with poor prognosis. Second, tumor site had a major influence. FoxP3+ T cells were associated with poor prognosis in hepatocellular cancer and generally good prognosis in colorectal cancer, whereas other cancer types were inconsistent or understudied. We conclude that FoxP3+ T cells have heterogeneous properties that can be discerned by the use of additional markers. Furthermore, the net biologic effects of FoxP3+ T cells seem to depend on the tumor site, perhaps reflecting microenvironmental differences. Thus, depletion of FoxP3+ T cells might enhance tumor immunity in some patient groups but be detrimental in others. Clin Cancer Res; 18(11); 3022–9. ©2012 AACR.

CD20+ tumor-infiltrating lymphocytes have an atypical CD27- memory phenotype and together with CD8+ T cells promote favorable prognosis in ovarian cancer.

Purpose: Tumor-infiltrating lymphocytes (TIL), in particular CD8+ T cells and CD20+ B cells, are strongly associated with survival in ovarian cancer and other carcinomas. Although CD8+ TIL can mediate direct cytolytic activity against tumors, the role of CD20+ TIL is poorly understood. Here, we investigate the possible contributions of CD20+ TIL to humoral and cellular tumor immunity. Experimental Design: Tumor and serum specimens were obtained from patients with high-grade serous ovarian cancer. CD8+ and CD20+ TIL were analyzed by immunohistochemistry and flow cytometry. Immunoglobulin molecules were evaluated by DNA sequencing. Serum autoantibody responses to the tumor antigens p53 and NY-ESO-1 were measured by ELISA. Results: The vast majority of CD20+ TIL were antigen experienced, as evidenced by class-switching, somatic hypermutation, and oligoclonality, yet they failed to express the canonical memory marker CD27. CD20+ TIL showed no correlation with serum autoantibodies to p53 or NY-ESO-1. Instead, they colocalized with activated CD8+ TIL and expressed markers of antigen presentation, including MHC class I, MHC class II, CD40, CD80, and CD86. The presence of both CD20+ and CD8+ TIL correlated with increased patient survival compared with CD8+ TIL alone. Conclusions: In high-grade serous ovarian tumors, CD20+ TIL have an antigen–experienced but atypical CD27− memory B-cell phenotype. They are uncoupled from serum autoantibodies, express markers of antigen-presenting cells, and colocalize with CD8+ T cells. We propose that the association between CD20+ TIL and patient survival may reflect a supportive role in cytolytic immune responses. Clin Cancer Res; 18(12); 3281–92. ©2012 AACR.

A novel spliced variant of the TIN2 shelterin is present in chronic lymphocytic leukemia

The shelterin proteins play important roles in telomere maintenance and genome stability. These proteins have been found to be mutated in many cancers including CLL. Herein, we demonstrate here the presence of a novel spliced isoform of TIN2S in chronic lymphocytic leukemia (CLL), related to deletion of exon 2 in the TIN2 gene. The expressions of spliced TIN2S mRNA varied widely in CLL and there was an inverse relationship between the mRNA levels of full-length TIN2S and the spliced moiety. Small amounts of spliced TIN2S were also observed in normal B cells but not in T cells. Spliced TIN2S appeared dysfunctional, as immunoprecipitation studies showed the typical association of TRF2 and TIN2 in normal lymphocytes but not in CLL cells. Moreover, whereas TRF2 localized to the nucleus in normal lymphocytes, it was present in both nuclei and cytoplasm in CLL cells. The levels of spliced TIN2S increased with age and in 3 of 8 patients increased over time. The presence of the spliced variant failed to be related to telomere length in CLL suggesting other functions for this protein. Further studies are required to determine the etiology and biological significance of this unique spliced TIN2S variant.

Abstract 2850: Role of shelterin proteins for telomere shortening in chronic lymphocytic leukemia

Telomeres are important in maintaining the integrity of the chromosomes. A series of shelterin proteins (TRF1, TRF2, RAP1, POT1a and b, TIPP1 and TIN2), are involved in the maintenance of telomeres. In chronic lymphocytic leukemia (CLL), the telomeres are shorter than in normal B cells, with very short telomeres being associated with poor survival. In the present study, we demonstrate that the leukemia cell telomere length shortens over time were CLL are followed over year, indicating that telomere shortening in these cells is an ongoing process. When CLL cell metaphases were examined by Q-FISH, telomere length shortening was found to affect all chromosomes equally. To determine whether these telomere changes were related to alterations in the shelterin complex, protein levels of the six shelterins were determined in CLL cells and normal pooled B cells. In general, apart from TIN2, the shelterin protein levels were increased in CLL cells and TRF2 levels were higher in IgVH unmutated cells than in mutated cells. TIN2 protein levels were dramatically reduced in CLL cells as compared to normal B cells and an alternative isoform of TIN2 was observed, both in IgVH mutated and unmuted cases. TIN2 targeted RT-PCR followed by sequencing revealed a deletion of the entire exon 2, totaling 105 bp. This abnormality appeared in monoclonal B-cell lymphocytosis (MBL) indicating that this was an early event. In addition, a subgroup of patients showed an increase in the spliced form of TIN2, when followed over time, suggesting that this might provide the cell with a survival advantage. A protein translated from the spliced variant RNA is expected to be deleted for 35 amino acids at the N-terminus, which is the binding site for TRF2. As TIN2 maintains telomere structure by forming a complex of TRF1 and TRF2 with the telomere, this should not occur with the splice variant. Indicating a pathological role of spliced TIN2 in CLL, TRF2 was localized in both the nuclei and cytoplasm in CLL cells compared to localization only to the nucleus in normal B cells. To confirm the function of the spliced TIN2, we have overexpressed the both full length and spliced clones of TIN2 in HEK293 cells and are presently determining the localization of TRF2 and the ability to form a TRF2/TIN2 complex. Thus, these studies demonstrate a unique splice variant of TIN2 in CLL, which potentially may influence the formation of the shelterin complex and contribute to telomere shortening in this disease. Citation Format: Ganchimeg Ishdorj, Sara EF Kost, Yunli Zang, Spencer B. Gibson, James B. Johnston. Role of shelterin proteins for telomere shortening in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2850.

Abstract 281: Bendamustine has the biochemical properties of an alkylating agent that synergizes with nucleoside analogues in chronic lymphocytic leukemia

Bendamustine (BEN) is a promising new treatment for chronic lymphocytic leukemia (CLL) and may function as an alkylating agent (eg, chlorambucil, CLB) while sharing structural similarities to a nucleoside analogue (eg, fludarabine, FLU). Enhanced cell death has been observed with the combination of BEN and FLU in primary CLL cells in vitro, but both BEN and FLU are marrow-toxic, potentially limiting the clinical value of this combination. While the nucleoside analogue pentostatin (PEN) is active in CLL and less marrow-toxic than FLU, it is unknown whether it produces synergy when combined with BEN. PEN acts by inhibiting adenosine deaminase, resulting in the accumulation of deoxyadenosine (dADO), which is toxic to CLL cells. In the present study, we evaluated the activity of BEN against CLL cells in vitro, as compared to CLB, FLU or dADO/PEN, alone and in combination. Cell death was assessed using annexin V/7-ADD staining and the MTT assay. We observed that BEN, CLB, FLU, or dADO/PEN induced apoptosis, the extent of which was time- and concentration-dependent. In general, cross-resistance was observed to all agents, with previously treated patients or those with a del 17p being most resistant. Enhanced cell killing was seen when combining BEN or CLB with FLU or dADO/PEN, with the extent of synergy being similar for FLU or dADO/PEN. To correlate mechanism of cell death as a result of treatment with these agents, we analyzed the expression of death receptors (DR4 and DR5) and mitochondrial stress (DICO6 and DHE). An increase in DR5 (but not DR4) surface expression, loss of mitochondrial membrane potential, and increased reactive oxygen species production was observed following treatment indicating that all agents induced death through the death receptor and mitochondrial apoptotic pathways. However, using γH2AX and the alkaline comet assay, FLU produced a greater number of DNA double-strand breaks (DSB) than BEN or CLB. Thus, all these agents induce apoptosis through the mitochondrial and death receptor pathways with cross-resistance being observed in most cases. BEN is more similar to CLB in producing fewer DNA DSBs than FLU and demonstrating synergy when combined with FLU or dADO/PEN. The latter observation suggests that combining BEN with PEN might be useful clinically. Citation Format: Sara EF Kost, Eric DJ Bouchard, Elise LaBossiere, Xibiao Ye, Michelle L. Queau, William S. Liang, Versha Banerji, Spencer B. Gibson, Sachin Katyal, James B. Johnston. Bendamustine has the biochemical properties of an alkylating agent that synergizes with nucleoside analogues in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 281.

Abstract 2555: The mechanism of action of bendamustine alone or in combination with nucleoside analogs in chronic lymphocytic leukemia

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Bendamustine (BEN) is a promising new treatment for chronic lymphocytic leukemia (CLL) and is believed to function as an alkylating agent (eg, chlorambucil, CLB) while sharing structural similarities to a nucleoside analogue (eg, fludarabine, FLU). Enhanced cell death has been observed with the combination of BEN and FLU in primary CLL cells in vitro, likely due to the inhibition of repair of BEN DNA cross-linking by FLU. However, both BEN and FLU are marrow-toxic, limiting the use of this combination in the clinic. The nucleoside analog, pentostatin (PEN), is an adenosine deaminase inhibitor that causes the accumulation of deoxyadenosine (dADO) and is less marrow-suppressive than FLU. However, the cytotoxic effectiveness of dADO/PEN in combination with BEN is not known. Flow cytometry analysis using annexin V/7-ADD and the MTT assay were performed to determine the effectiveness of BEN treatment in primary CLL cells as compared to or in combination with CLB, FLU, or dADO/PEN at varying drug concentrations and time points. Using the MTT assay, there was a linear relationship between cytotoxicity and drug concentration, similar to that seen with CLB. In vitro treatment of CLL cells with BEN, CLB, FLU, or dADO/PEN induced apoptosis, the degree being time- and concentration-dependent. However, the sensitivity of CLL cells to BEN or CLB varied, suggesting different mechanisms of action. Enhanced cell kill was seen with the combination of BEN or CLB with FLU or dADO/PEN, with the extent of increased cytotoxicity being similar for FLU or dADO/PEN. Cell death mechanisms were analyzed via staining for surface expression of death receptors (DR4 and DR5) and mitochondrial stress (DICO6 and DHE). γ-H2AX staining was used to measure DNA double-strand breaks (DSBs) and the alkaline comet assay was used to measure both DNA DSBs and single-strand breaks (SSBs). An increase in DR4 and DR5 surface expression, loss of mitochondrial membrane potential, and increased reactive oxygen species production was observed with all agents. There was an increase in DNA SSB and DSB following FLU, as compared to BEN and CLB, while a combination of BEN and FLU further increased the number of breaks (especially SSBs) compared to FLU alone. Thus, BEN has the properties of an alkylating agent, rather than a nucleoside analog, but has a different spectrum of antitumor activity to CLB. BEN induces apoptosis through both the death receptor and mitochondrial pathways. Increased cell kill is seen on combining BEN with dADO/PEN suggesting that this might be a useful combination in the clinic. Citation Format: Sara E.F. Kost, Eric D.J. Bouchard, William S. Liang, Versha Banerji, Spencer B. Gibson, Sachin Katyal, James B. Johnston. The mechanism of action of bendamustine alone or in combination with nucleoside analogs in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2555. doi:10.1158/1538-7445.AM2015-2555

Abstract B76: CD8+FoxP3+ T cells: A new player in the immune response to ovarian cancer.

Introduction: Tumor-infiltrating lymphocytes (TIL) are an important prognostic indicator in high-grade serous ovarian carcinoma (HGSC). Certain types of TIL (in particular CD8+ effector T cells) predict better outcomes, whereas others (most notably CD4+CD25+FoxP3+ regulatory T cells or Tregs) predict worse outcomes. An unconventional subset of CD8+FoxP3+ T cells has been widely reported in autoimmunity. While the functional significance of CD8+FoxP3+ TIL remains poorly understood, in a murine tumor model they were associated with effective anti-tumor responses. Hypothesis: CD8+FoxP3+ TIL are present in HGSC and correlate with effector TIL and increased patient survival. Experimental Design: Multi-parameter flow cytometry and immunohistochemistry (IHC) were performed on cohorts of 12 and 45 primary HGSC specimens, respectively, to enumerate and locate CD8+FoxP3+ TIL. For IHC, intraepithelial versus stromal location was determined by staining adjacent sections for the epithelial marker cytokeratin. Multi-color staining was resolved using the Nuance™ multispectral imaging system in conjunction with Metamorph software. Survival analysis will be performed by staining a cohort of 200 cases of HGSC. Results: By flow cytometry, CD8+FoxP3+ cells were found in variable proportions between HGSC cases, representing 1.3 - 7.9% of CD8+ TIL and 1.3 - 25.8% of FoxP3+ TIL. By IHC, CD8+FoxP3+ TIL were found together with CD8+FoxP3- and CD4+FoxP3+ TIL in epithelial and stromal tumor regions and represented up to 19% of the FoxP3+ population. Interestingly, the majority of CD8+FoxP3+ TIL were also found to be TIA-1+KI67-, consistent with an active role in the anti-tumor response. Conclusions: CD8+FoxP3+ TIL are a component of the host immune response to HGSC. Survival analysis is ongoing and will determine whether CD8+FoxP3+ T cells enhance or inhibit tumor immunity. Improved understanding of this new TIL subset will inform future immunotherapy strategies for this challenging malignancy. Citation Format: Sara E.F. Kost, Ronald J. deLeeuw, Brad H. Nelson. CD8+FoxP3+ T cells: A new player in the immune response to ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B76.

Abstract A88: Identification of factors modulating sensitivity to T cell infiltration in the breast cancer setting via next-generation sequencing.

Clinical and pre-clinical trials have established that killer (CD8+) T cells can recognize and destroy tumors. The challenge of our time is to address barriers to T cell infiltration into solid tumors and thereby make cancer immunotherapy a powerful, broadly applicable treatment modality. Bilateral breast cancer represents a unique opportunity to identify those proteins that modulate immune cell entry into solid tumors. Bilateral synchronous breast cancer is when tumors arise independently in separate breasts of the same patient at the same time. Similar to the powerful use of “twin studies” in epidemiology, bilateral tumors provide an opportunity to explore two independent tumorigenic events against a common genetic (and immunological) background. We hypothesise that the sensitivity of breast cancers to T cell infiltration can be predicted on the basis of their molecular profiles. To assess this question, a cohort of bilateral and unilateral breast cancer tissues has been assembled in collaboration with CTRNet-affiliated biobanks. Our objective was to identify 10 tumor pairs with differential T cell infiltration. Here we present the novel protocol for multi-colour brightfield immunohistochemistry (IHC) which was used to quantitate multiple lymphocyte subsets in these precious specimens at the same time. A workflow for automated image collection is presented, which is geared to the analysis of whole-tumour sections. Our results demonstrate that bilateral breast tumors often show differential lymphocyte infiltration despite being exposed to the same level of systemic immunity. Paired tumors will be subjected to whole transcriptome sequencing and lymphocyte receptor spectratyping to identify proteins that distinguish infiltrated from non-infiltrated tumors. Thus, patients with bilateral breast cancer have provided a powerful, translationally-relevant opportunity to study local barriers to T cell infiltration in solid tumors. Citation Format: Sally Amos, Ron de Leeuw, Nikita Kuklev, Juzer Kakal, Sindy Babinsky, Katy Milne, Sara Kost, Doug Freeman, Rob Holt, Peter Watson, Brad Nelson. Identification of factors modulating sensitivity to T cell infiltration in the breast cancer setting via next-generation sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A88.

Abstract B88: Clinical significance of FOXP3+ T cell heterogeneity in ovarian cancer.

Background: CD8+ tumor-infiltrating lymphocytes (TIL) are associated with survival in ovarian cancer. A second subpopulation of TIL – defined by FoxP3 expression – has been reported to inhibit tumor immunity, resulting in decreased patient survival. However, recent studies by our lab and others have challenged this paradigm by showing that FoxP3+ T cells, in some patient cohorts, are associated with favorable prognosis. Hypothesis: FoxP3 expression encompasses heterogeneous populations of effector and regulatory T cells that vary in proportion between patients and provide differential prognostication. Methodology: We used multi-parameter flow cytometry to assess CD25, CD28, CD39, CD45RO, CD103, CD127, CTLA-4, CCR4, CCR7, GITR, Helios, ICOS, IFN-gamma, and PD-1 in CD3+CD4+CD8-FoxP3+ TIL in 12 patients with high-grade serous ovarian cancer (HGSC). CD4 and CD8 expression within FoxP3+ TIL was assessed by four channel multi-parameter immuno-histochemistry (mIHC). CD8-FoxP3+ TIL were assessed for CD25 and CD39 expression by mIHC. Survival analysis will be assessed via Kaplan-Meier plots, log-rank tests and Cox regression analyses. Results: Consistent with prior reports, flow cytometry revealed that FoxP3 was expressed predominantly by CD4+ TIL. All CD4+FoxP3+ cells also expressed CD28, CD45RO, ICOS and CTLA-4, while they lacked expression of CD103 and IFN-gamma. However, we observed highly varied expression of regulatory T cell markers, including CD25, CD39, and GITR. Furthermore, on average, 4% (range 0-25%) of FoxP3+ TIL were CD8+. TCR spectratyping revealed similar TCR Vβ usage patterns between FoxP3- and FoxP3+ TIL. Although mIHC confirmed the variable expression of CD39 in CD8-FoxP3+ TIL, the expression of CD39 by a portion of HGSC tumors eliminated our ability to assess the clinical significance of this subset. The clinical significance of CD25 expression in CD8-FoxP3+ TIL is currently being analyzed in 245 cases of HGSC by mIHC. Conclusions: FoxP3+ TIL demonstrate profound phenotypic heterogeneity within and between HGSC patients. FoxP3- and FoxP3+ TIL appear to be clonally related, suggesting their distinct phenotypes reflect microenvironmental influences rather than developmental origin or antigen specificity. The variable expression of CD25 and CD39 may be of importance in defining clinically important FoxP3+ TIL subsets. Further study of FoxP3+ TIL subpopulations in HGSC is required to better understand their contributions to tumor immunity and patient survival. Citation Format: Ronald J. deLeeuw, Sara E. Kost, Brad H. Nelson. Clinical significance of FOXP3+ T cell heterogeneity in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B88.